1) Are you using 33342 or 33258? Concentration?
2) Are you using log or lin acquisition for the Hoechst?
3) Do you use PI exclusion? How about 7-AAD? Concentration?
4) How long do you expose the cells to the dyes before reading? Temp?
My concerns mostly center around the third question... the 7-AAD signal
just doen't look like I expect it should. Several labs I work with have
been using the following protocol with various cell types, including
chondrocytes, HL-60, and fresh human T cells:
1.Resuspend at least 0.5 x 10^6 cells in 12x75mm tubes at a
concentration of 10^6 cells/ml.
2.Add Ho 33258 at a final concentration of 1ug/ml to samples.
3.Incubate the samples in a 37 C waterbath for 7 minutes.
4.Remove samples from the waterbath and immediately place on ice.
5.Add 7-AAD at a final concentration of 1ug/ml to samples.
6.Incubate on ice for 10 minutes.
7.Analyze samples, with log amplification for both Hoechst and 7-AAD.
I almost never see any Hoechst+/7-AAD- cells, no matter what the
treatment. Only double negatives and double positives. If I do an
Annexin/7-AAD expeiment, I see Annexin+/7-AAD- cells, suggesting cells
that are clearly apoptotic, with intact membranes. Aren't these the
type of cell that would accumulate Hoechst but exclude 7-AAD?
One thing to consider is that I am reading 7-AAD off of two differnet
PMTs, and exciting with two different lasers on our FACStar+. I get
good sensitivity & low CVs when lloking at alignment beads in either
configuration, but I still suspect the different 7-AAD staining profiles
may be related to the different configurations.
For Annexin experiments, all excitation is off of the front I90, in
488nm/250mW. Annexin is measured on FL-1 with a 530BP and a 560DS, and
7-AAD is being measured on FL3-1 with a 660BP and the 610DS split
mirror.
For Hoechst experiments, scatter and Hoechst are being excited by the
front I90 in UV/50mW, and 7-AAD is being excited by the rear I70 in
488nm/250mW. Hoechst is being measured on FL1 with a 424BP and a 505DS,
and 7-AAD is being measured on FL4 with a 660BP, 640DL, and the 610DS
split mirror.
Any feedback would be greatly appreciated.